The present invention relates to the demonstration of immuno-redox activities of heterologous SODs (HSDs) and to a novel therapeutic use, as well as to a method for selecting said HSDs.
Under the normal conditions of oxygen pressure, oxygen gives rise to reactive oxygen (hydrogen peroxide and free radicals such as superoxide anions, hydroxyl radicals or nitric oxide), which is rapidly destroyed by cells which have at least three major endogenous mechanisms for autoregulation of the reactive oxygen produced (antioxidant substances, metal chelaters, detoxifying enzymes).
Conversely, if these mechanisms are affected, or if the production of reactive oxygen, and in particular free radicals, is too great, an oxidative stress and the appearance of a pathological state results therefrom.
Superoxide dismutases or SODs, which form a class of metalloproteins containing iron, copper, zinc or manganese, are enzymes which are capable of inducing the dismutation of superoxide anions to protect cells against the toxicity of these superoxide radicals (O2xe2x88x92).
Superoxide anions generally form when molecular oxygen acquires an additional electron, which happens when oxygen is subjected to ionizing radiation. This anion has a short life, and is converted into hydrogen peroxide by SOD (dismutation). This dismutation generates hydrogen peroxide which is also a cellular oxidant. To respond to the rise in hydrogen peroxide concentrations, cells increase the activity of enzymes involved in eliminating hydrogen peroxide, i.e. catalase and glutathione peroxidase, in accordance with the following scheme:
2O2xe2x88x92+2H+xe2x86x92H2O2+O2xe2x80x83xe2x80x83superoxide dismutase
2H2O2xe2x86x92O2+2H2Oxe2x80x83xe2x80x83catalase
xe2x80x83ROOH+2GSHxe2x86x92ROH+H2O+GSSGxe2x80x83xe2x80x83glutathione peroxidase.
In the absence of sufficient amounts of catalase and of glutathione peroxidase, and in the presence of small amounts of iron, which result in particular from cellular lesions, a conversion is obtained of hydrogen peroxide into an even more toxic compound, hydroxyl radicals (Fenton reaction), in accordance with the following scheme:
O2xe2x88x92+Fe3+xe2x86x92Fe2++O2
Fe2++H2O2xe2x86x92Fe3++OHxe2x88x92OHo
O2xe2x88x92+H2O2xe2x86x92O2+OHxe2x88x92+OHo
Since oxygen-derived free radicals appear to be involved in many disorders, the use of SOD in therapeutics has thus been advocated in pathologies induced by reactive oxygen such as inflammatory states (inflammatory arthropathies, for example) (R. NORDMANN et al., Cah. Nutr. Diet., 1991, 26, 6, 398-402); in lung diseases, and more particularly broncho-pulmonary dysplasia, or in other toxic conditions linked to the presence of oxygen in considerable amounts (central nervous system, ischaemia, nonvascular gastrointestinal disorders, ocular disorders (locally in the anterior chamber of the eye) or combating the undesirable effects of anti-cancer treatments) SOD has also been proposed, with a greater or lesser degree of success (Greenwald R. A., Free Radical Biol. Med., 1990, 8, 201-209).
Three distinct types of SOD have been described, which fall within two distinct developing families;
SOD containing copper and zinc, which is usually located in the cytosol of eukaryotic cells, in the extracellular fluid of mammals and in some bacteria;
SOD containing manganese (MnSOD) or iron (FeSOD), which is usually located in prokaryotes or in mitochondria (MnSOD);
SOD containing iron (FeSOD), which is located in anaerobic bacteria and prokaryotes.
Among the SODs tested, it is those which exhibit a sustained half-life and a low incidence of accidents of immunological nature which have preference; mention may be made in particular of Cu/Znxe2x80x94SOD of bovine origin (homodimer which catalyses the dismutation of the superoxide radical), Mnxe2x80x94SOD of E. coli, Fexe2x80x94SOD, liposomal SODs, polyethylene glycol-conjugated SODs, SOD polymers or copolymers, recombinant human CuZnxe2x80x94SOD and Mnxe2x80x94SOD, as well as SODs of plant origin.
In various inflammation inhibition tests, heterologous SODs are found to exhibit significantly greater anti-inflammatory activity than homologous SODs.
For example:
in a rat model in which an inflammation is induced with carrageenans, the anti-inflammatory activity of various SODs is as follows: Mnxe2x80x94SOD of E. coli greater than bovine Cuxe2x80x94SOD greater than human Cuxe2x80x94SOD greater than yeast Cuxe2x80x94SOD greater than plant Cuxe2x80x94SOD; in this case, rat Cuxe2x80x94SOD exhibits significant pro-inflammatory activity;
in a rat model in which an inflammation is induced with adriamycin, the anti-inflammatory activity of various SODs is as follows: bovine Cuxe2x80x94SOD greater than Mnxe2x80x94SOD of E. coli; rat homologous Cuxe2x80x94SOD is totally inactive, whereas yeast Cuxe2x80x94SOD generates a pro-inflammatory response.
The applicant has found, unexpectedly, that, besides a dismutase activity, heterologous SODs (HSDs) possess an immuno-redox activity which it is possible to dissociate from the dismutase activity.
For the purposes of the present invention, xe2x80x9cimmuno-redox activityxe2x80x9d is intended to mean a stimulation of the production of endogenous SOD, catalase and glutathione peroxidase.
Besides the fact that such heterologous SODs with essentially immuno-redox activity do not induce a pro-inflammatory reaction, they induce the production of endogenous SOD and stimulate the production of catalase and of glutathione peroxidase. Such SODs thus increase nonspecific defences which protect against cell and organ degeneration.
A subject of the present invention is the use of a plant heterologous SOD with essentially immuno-redox activity for preparing a medicinal product intended for treating diseases in which cell and organ degeneration is observed.
Such heterologous SODs which increase nonspecific defences protect cells from degeneration; they thus constitute an antidegeneration medicinal product of choice, whatever the origin of the degeneration: degenerative pathology such as radiation-induced aftereffects, Parkinson""s disease, Alzheimer""s disease, etc., degeneration induced by an infectious agent (AIDS, bilharzia, post-infectious cirrhosis (hepatitis C)) or iatrogenic degeneration (medicinal detoxification action).
According to an advantageous embodiment of said use, said SOD with essentially immuno-redox activity is a nitrated SOD; such an SOD has lost its dismutase activity, whereas it conserves its immuno-redox activity.
It has the advantage of avoiding any risk of pro-inflammatory activity linked to the dismutase activity and to the production of an excess of hydrogen peroxide, when the induction of catalase and glutathione peroxidase production is too weak to satisfy the demand.
Surprisingly, SODs according to the invention (HSDs), which no longer, or practically no longer, exhibit dismutase activity, but which have conserved their immuno-redox activity, used as a medicinal product in humans, stimulate the production of endogenous SOD, as well as the production of catalase and of glutathione peroxidase.
According to another advantageous embodiment of said use, said plant heterologous SOD is in particular derived from melon.
According to another advantageous embodiment of said use, said SODs are used for preparing a medicinal product intended for treating degenerative diseases which are selected from the group consisting of neurodegenerative diseases, cirrhosis, lentivirus infections, parasite infections and iatrogenic diseases (medicinal detoxification).
A subject of the present invention is also a method for selecting a plant heterologous SOD with essentially immuno-redox activity, characterized in that it comprises:
(a) measuring the dismutase activity of an SOD, or of a modified SOD,
(b) selecting SODs without dismutase activity or having a dismutase activity which is reduced by at least a factor of 10, and
(c) measuring the immuno-redox activity of the SODs selected in (b) in a cellular system in which expression of endogenous SOD is inhibited.
According to an advantageous embodiment of said method, step (a) for measuring the dismutase activity is carried out by reduction of ferricytochrome c.
According to another advantageous embodiment of said method, the system according to step (c) consists of cells expressing the Tat protein of HIV-1.